Bifidobacteria uses insoluble resistant starch (RS) fractions as prebiotics in the colon, consequently improving human intestinal health. In this study, various α-1,6 linkage ratios of insoluble α-glucans were synthesized by amylosucrase from Neisseria polysaccharea and glycogen branching enzyme from Rhodothermus obamensis to investigate the effect of branching patterns on Bifidogenic property. Different ratios (0%–4%) of α-1,6 branched insoluble α-glucans with α-1,4 linked backbones were effectively synthesized. Additionally, the proportions of RS fraction in the synthesized α-glucans were 69.5%–81.9% after the cooking process, which are higher amounts than amylomaize VII (36.8%). As various Bifidobacteria used the insoluble branched α-glucans , the structure with 2.5% of 1,6 linkage showed the highest consumption yield, more than 70%, among all tested strains. Therefore, the insoluble α-glucans with different α-1,6 linkage ratios can be applied as prebiotic ingredients to modulate the Bifidobacterium species to enhance colon health and related outcomes. • Amylosucrase and glycogen branching enzyme was applied to synthesize α-glucans. •Insoluble α-glucans with different α-1,6 linkage ratios were successfully produced. •Insoluble branched α-glucans contain high amounts of resistant starch fraction. •Bifidobacterium species broadly used the insoluble α-glucans as prebiotics.